GenElute™ mRNA Miniprep Kits

Procedures such as cDNA synthesis, expression profiling, and others, require separation of mRNA from the vastly more abundant rRNA and tRNA. The GenElute mRNA Kits provide convenient procedures for isolating polyadenylated mRNA from previously prepared total RNA, or directly from mammalian cells and tissues.

For direct mRNA preparation, cells or tissues are disrupted with SDS/proteinase K digestion to release RNA and eliminate RNases. Both kit types use oligo dT30 covalently linked to 1 µm polystyrene beads to capture polyadenylated mRNA by hybridization. The polystyrene beads remain suspended during hybridization, eliminating the need for mixing or rocking, as is common for cellulose or magnetic particles. Polystyrene was also chosen because oligo (dT) polystyrene beads yield cleaner mRNA with fewer stringent washing steps than does the more commonly used oligo (dT) cellulose (2 or 3 wash steps versus 10 or more). With the GenElute kits, mRNA-bead complexes are washed on a microcentrifuge spin filter, and eluted into 10 mM Tris-HCl, pH 7.5. mRNA prepared with either kit is suitable for a variety of downstream applications such as Northern Blotting (Figure 4), Expression Array or Chip Hybridizations, and cDNA Synthesis and Library Construction.

Figure 1. Comparison of the preparation time, to isolate mRNA from total RNA or direct from cells or tissues, using GenElute mRNA Miniprep Kit and other commercially available kits. Each kit was prepared according to the procedure supplied by the vendor.

Figure 2. Total RNA was prepared from Hek293 cells by a silica-binding method, using TriReagent®. mRNA was subsequently prepared from total RNA preparations using commercially available kits, following the procedures supplied by each vendor. RNA yield was measured using the RiboGreen™ RNA Quantitation Kit (Molecular Probes).

Figure 3. mRNA was prepared from total RNA as in Figure 2, and twenty percent of each preparation was analyzed by Northern blot hybridization with 32P-labeled probes. Hybridization to human HGPRT, human p53, and mouse p53 mRNAs were quantified with a Perkin Elmer Instant Imager.

Figure 4.Northern blot comparison of mRNA prepared with GenElute mRNA kits from other suppliers. Duplicate mRNA samples were prepared from 5 x 106 Hek293 cells (top left panels) or 25-35 mg mouse liver (top right panels) with Sigma's GenElute Direct mRNA Miniprep Kit (S) or with another commercially available direct mRNA miniprep kit (A, D, I, P, & Q). An optional release and rebind procedure (described in the Manual was included for preparation S+: RNA was hybrid-captured onto oligo dT beads, released into fresh lysis solution, and re-captured onto the same beads before washing and eluting mRNA. The release and rebinding steps were omitted for S- & Sx2 preparations. Sx2 preparations were repurified after the final elution with fresh oligo dT beads (as described in the Manual. Equal portions of each mRNA preparation, equal to the amount from 1 x 106 cells or 10 mg liver, were evaluated by Northern blot hybridization with 32P-labeled RNA probes for p53, hypoxanthine-guanine phosphoribosyl transferase (HGPRT), c-myc, and/or glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA.

For the bottom panels, total RNA was first prepared from Hek293 cells and from mouse liver. Duplicate mRNA samples were then prepared from 100 µg of these total RNA preparations with Sigma's GenElute mRNA Miniprep Kit (S) or with another commercially available mRNA miniprep kit (Q, A, I, P, D). Samples Sx2 were repurified after elution. Twenty percent of each preparation was evaluated by Northern blot as above. In lanes T, 5 and 2 µg of the original total RNA from cells or 10 and 5 µg of total RNA from liver were analyzed for comparison.

Genelute Kit (Product No. MRN10) was used to extract the mRNA from chilli leaves
Lane 1 & 7 has wide range marker D7058, Lane 2 & 3 mRNA preparation was shown to have no DNA contamination with no DNAse treatment as well as with Dnase treatment, lane 3 & 4 are PCR positive controls using 1 µg and 100 ng of leaf DNA as templet, Lanes 9 and 10 are showing results of RbcL geneRT PCR amplification mRNA without any DNAse or with DNAse treatment. The size of DNA and RNA amplicon is 386 bp.

Genelute Kit (Product No. MRN10) was used to extract the mRNA from maize leaves
Lane 1 & 7 has wide range marker D7058, Lane 2 & 3 mRNA preparation was shown to have no DNA contamination with no DNAse treatment as well as with Dnase treatment, lane 3 & 4 are PCR positive controls using 1 µg and 100 ng of leaf DNA as tempelets, Lanes 9 and 10 are showing RbcS gene RT PCR amplification of mRNA with out any DNAse or with DNAse treatment. The size of DNA amplicon is 601 bp and RNA amplicon is 438 bp.

Genelute Kit (Product No. MRN10) was used to extract the mRNA from sugarcane leaves
Lane 1 & 7 has wide range marker D7058, Lane 2 & 3 mRNA preparation was shown to have no DNA contamination with no DNAse treatment as well as with Dnase treatment, lane 3 & 4 are PCR positive controls using 1 µg and 100 ng of leaf DNA as tempelets, Lanes 9 and 10 are showing Actin gene RT PCR amplification of mRNA with out any DNAse or with DNAse treatment. The size of DNA amplicon is ~2030 bp and RNA amplicon is 1057 bp.